Background Smyd1b is a member of the Smyd family that takes

Published December 27, 2016

Background Smyd1b is a member of the Smyd family that takes on a key part in sarcomere assembly during myofibrillogenesis. could methylate histone H3 proteins [4]. Consistent with its potential function in transcriptional rules Smyd1 is definitely in the beginning localized in the nucleus of C2C12 myoblasts [5]. studies have exposed that represses gene transcription inside a histone deacetylase (HDAC) dependent fashion [3]. However it has been reported that Smyd1 undergoes a nucleus to cytoplasm translocation during myoblast differentiation into myotubes [5] suggesting that Smyd1 may have additional function in the cytoplasm. A better characterization of Smyd1b localization is critical for the mechanistic understanding of Smyd1b function in regulating muscle mass cell differentiation. With this study we analyzed the subcellular localization Smyd1b_tv1 and Smyd1b_tv2 during muscle mass development in zebrafish embryos as well as with adult skeletal muscle tissue. The data showed that Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes of zebrafish embryos at the early stage. However in adult myofibers of late stage embryos a sarcomeric localization was obvious for Smyd1b_tv1 and Smyd1b_tv2 although Smyd1b_tv2 appeared to be weaker. Two times immunostaining with M- or Z-line markers exposed that Smyd1b_tv1 was localized within the M-line of sarcomeres. The strong M-line localization requires Phe223 and Ser225 located within the Smyd1b_tv1-specific 13 aa insertion. Mutation of Phe223 or Ser225 to alanine significantly reduced the sarcomeric localization 3-Cyano-7-ethoxycoumarin of Smyd1b_tv1. In contrast replacing Ser225 with threonine experienced no effect on the Smyd1b_tv1 sarcomeric localization Results Characterization of Smyd1b_tv1 and Smyd1b_tv2 subcellular localization during muscle mass development in zebrafish embryos Earlier studies have shown that Smyd1 undergoes a nucleus to cytoplasm translocation during C2C12 myoblast differentiation [5]. It is not clear whether the two isoforms Smyd1b_tv1 and Smyd1b_tv2 from option splicing 3-Cyano-7-ethoxycoumarin have related or unique subcellular localization in muscle mass cells during muscle mass development. To better understand Smyd1b function in myofibril assembly we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 during muscle mass development using transgenic zebrafish models that indicated a myc-tagged Smyd1b_tv1 3-Cyano-7-ethoxycoumarin or Smyd1b_tv2 under the control of its own promoter (Fig. 1). The results showed a dynamic subcellular localization of Smyd1b_tv1myc and Smyd1b_tv2myc during muscle mass development. In early stage embryos at 14 and 24 hpf Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblast and myotubes with little or no nuclear 3-Cyano-7-ethoxycoumarin localization (Fig. 2A-D). However as embryos develop into late stages a definite sarcomeric localization was recognized for Smyd1b_tv1 in differentiated myofibers at 27 hpf (Fig. 2E). The sarcomeric localization appeared earlier for Smyd1b_tv1 than Smyd1b_tv2 (Fig. 2F). The sarcomeric localization of Smyd1b_tv1myc was managed throughout early development (Fig. 2G I K). In contrast a poor sarcomeric localization of Smyd1b_tv2 could be recognized in zebrafish embryos at 72 hpf (Fig. 2L). Number 1 Generation of Smyd1b_tv1 and Smyd1b_tv2 by alternate splicing and building of the Smyd1b_tv1myc and Smyd1b_tv2myc transgenes. Number 2 Smyd1b_tv1myc and Smyd1b_tv2myc display dynamic localizations during muscle mass cell differentiation in zebrafish embryos. The timing of Smyd1b_tv1myc sarcomeric localization was compared with other sarcomeric proteins in trunk muscle tissue of the same stage zebrafish embryos. TRUNDD The results showed the sarcomeric localization of myosin weighty chain α-actin and α-actinin occurred before the Smyd1b_tv1 sarcomeric localization (Fig. 3). Solid and thin filaments as well as Z-lines were clearly structured in myofibers of zebrafish embryos at 24 hpf (Fig. 3A-C). In contrast there was little sarcomeric localization of Smyd1b-tv1myc in trunk muscle tissue of the same stage embryos except muscle mass pioneer cells in the myoseptum region representing the 1st group of muscle mass cells to differentiate in zebrafish embryos (Fig. 3D). Collectively these data show the sarcomeric localization of Smyd1b_tv1 occurred after that of myosin α-actin and α-actinin suggesting that although Smyd1b is required for myofibril assembly the sarcomeric localization of Smyd1b_tv1 was not required with this initial process. Number 3 The sarcomeric localization of Smyd1b_tv1 occurs after the sarcomere formation in myofibers of zebrafish embryos..